Peptide isolated from micrococcus varians and use thereof

ABSTRACT

An isolated peptide which in a microorganism, and particularly in Micrococcus varians, is a signal peptide which initiates expression of a bactericide composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.08/693,353, filed Aug. 6, 1996.

BACKGROUND OF THE INVENTION

The present invention relates to a bacteriocin, to a strain whichproduces this bacteriocin, to a process for preparing this bacteriocin,and to the use of this bacteriocin and/or a strain producing thisbacteriocin in the manufacture of foodstuffs and cosmetics.

STATE OF THE ART

Bacteriocins have been isolated from numerous Gram-positive andGram-negative bacteria. Bacteriocins are molecules which are essentiallyproteinaceous in nature and which possess a bactericidal effect and, forthis reason, a bacteriocin provokes an antagonistic reaction between thebacterium which produces it and one or more different bacterial species.Furthermore, the inhibition spectrum of a bacteriocin is often limitedto the species which are closely related to the bacterial species whichproduces it.

Bacteriocins have, in particular, been demonstrated in lactic acidbacteria. For example, EP 0643136 (Societe des produits Nestle)described the identification of two bacteriocins from Streptococcusthermophilus.

Similarly, a bacteriocin has been isolated from Lactococcus lactis (App.and Env. Microbio. 58, 279-284, 1992; J. of Bio. Chem. 268, 16361-16368,1993).

However, to date, no bacteriocin is known which is derived fromMicrococcus varians. Micrococcus varians is now much used within thefoodstuff sphere, in particular in fermentation of meat for the purposeof manufacturing delicatessen products such as salamis and sausages, forexample. It would, therefore, be very useful to have available abacteriocin-producing strain in order to combat pathogenic agents.

The object of the present invention is to respond to this need.

SUMMARY OF THE INVENTION

To the end of meeting the object stated above, the present inventionprovides a bacteriocin produced by Micrococcus varians, provides strainswhich produce the bacteriocin, provides a process for preparing thebacteriocin, provides nucleotide fragments which encode the bacteriocinand for preparing the bacteriocin which is fused to a signal peptide,and the present invention provides the amino acid sequence of thepeptide, which has been isolated.

The bacteriocin according to the present invention is a bacteriocin fromMicrococcus varians, and has agar well incubation inhibition testactivity against at least one of Lactobacillus, Streptococcus,Enterococcus, Listeria, Bacillus, Clostridia and Staphylococcus andexhibits the amino acid sequence SEQ ID NO:1 or any amino acid sequencediffering from the sequence SEQ ID NO:1 by one substitution, onedeletion and/or one insertion of from 1 to 4 amino acids.

Furthermore, any nucleotide fragment encoding this bacteriocin,including encoding amino acid sequences SEQ ID NO:1 and/or SEQ ID NO:3as set forth and identified below, and in particular the nucleotidefragment exhibiting the sequence SEQ ID NO:2 identified in the sequencelist below also comes within the scope of the present invention.

Further included in the present invention is an isolated peptide, thepeptide being a signal peptide for the bacteriocin and having aminoacids of the amino acid sequence minus 22 to minus 1 of sequence SEQ IDNO:3, as identified and set forth below, and which has a signal peptidein a microorganism initiates expression of the bacteriocin.

The strains according to the present invention are Micrococcus variansstrains which produce this bacteriocin, in particular the Micrococcusvarians strains CNCM I-1586 and CNCM I-1587.

In the process for preparing the bacteriocin according to the presentinvention, a Micrococcus varians strain which produces the bacteriocin,in particular the strain CNCM I-1586 or the strain CNCM I-1587, iscultured, in a medium and under conditions which are favourable forgrowth, so as to obtain a culture medium containing from 10⁷ to 10¹¹organisms of this strain per ml, after which the supernatant is isolatedfrom the resulting culture by separating the supernatant from thecultured cells to obtain a supernatant containing the bacteriocin, andto effect separation, the resulting culture is centrifuged and asupernatant extract containing the bacteriocin therefore obtained. Thesupernatant may be concentrated to obtain a concentrate comprising thebacteriocin, and the bacteriocin may be isolated from the supernatantand concentrate by dehydration to obtain a powder, and an isolated andpurified bacteriocin may be obtained from the supernatant andconcentrate and may be dehydrated.

Finally, the use of the Micrococcus varians bacteriocin according to theinvention comprises using its nucleotide sequence, as well as its signalsequence, and using the supernatant extract containing the bacteriocin,and a Micrococcus varians strain which produces the bacteriocin, forpreparing foodstuffs and cosmetics.

DETAILED DESCRIPTION OF THE INVENTION

In that which follows, the bacteriocin according to the presentinvention will be termed "variacin".

Within the meaning of the present invention, an arbitrary unit (au) isdefined as the inverse of the value of the largest dilution at which asample still exhibits a bactericidal effect in the test which is knownto the skilled person as the "agar well test".

Within the meaning of the present invention, the term "fragment" or "DNAfragment" is to be understood to mean a single-stranded ordouble-stranded DNA fragment which is partially or entirely coding andwhich can be synthesized, replicated in vitro by, for example, the knownpolymerase chain reaction method, or replicated in vivo in a bacteriumof the Escherichia coli type, for example.

Within the meaning of the present invention, a "homologous fragment" isunderstood to mean any fragment which only differs from the fragmentsaccording to the invention by the substitution, deletion or insertion ofa small number of bases. Within this context, two DNA fragments whichencode one and the same polypeptide, due to the degeneracy of thegenetic code, will, in particular, be regarded as being homologous. Thatfragment will also be regarded as being an homologous fragment whichexhibits more than 80% homology with the fragment according to theinvention. In this latter case, the homology is determined by the ratiobetween the number of bases in a homologous fragment and the number in afragment according to the invention.

Finally, within the meaning of the present invention, "homologousfragment" is also understood to mean any fragment which is able tohybridize with the fragments according to the present invention by theSouthern blot method (Sambrook et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, U.S.A., 1989, chapter 9.31to 9.58). Preferably, the hybridization is carried out under rigorous orstringent conditions so as to avoid non-specific hybridizations orhybridizations which are relatively unstable.

A proteinaceous factor, in this instance a bacteriocin possessing apowerful bactericidal effect, has been isolated from the strains CNCMI-1586 and CNCM I-1587. This bacteriocin, which is derived fromMicrococcus varians and which consequently exhibits the amino acidsequence SEQ ID NO:1, which is described in the sequence list below, hasbeen termed variacin.

In view of the interest afforded by variacin, the invention also relatesto any bacteriocin which possesses an amino acid sequence which differsfrom the sequence SEQ ID NO:1 by one substitution, one deletion and/orone insertion of from 1 to 4 amino acids. Thus, the said bacteriocin,which exhibits an amino acid sequence differing from the sequence SEQ IDNO:1 by one substitution, one deletion and/or one insertion of from 1 to4 amino acids, may have an inhibition spectrum for a bacterial genus orbacterial species which is wider than that of the said variacin, forexample.

It has also been possible to select a chromosomal nucleotide fragmentencoding the variacin according to the invention from the two strainsCNCM I-1586 and CNCM I-1587. The said fragment exhibits the sequence SEQID NO:2 given in the sequence list below.

In view of the interest afforded by the present invention, the inventionalso relates to any nucleotide fragment which encodes the variacinaccording to the present invention, in particular to nucleotidefragments which are homologous to or which hybridize with the sequenceSEQ ID NO:2.

In particular, the invention relates to nucleotides 88 to 153 of thesequence SEQ ID NO:2, encoding the signal peptide of the variacin, tonucleotides 154 to 228 of the sequence SEQ ID NO:2, encoding thesecreted variacin according to the present invention, and/or tonucleotides 88 to 228 of the sequence SEQ ID NO:2, encoding thebacteriocin fused to its signal peptide.

The fragment encoding the secreted variacin may be advantageously usedto express the variacin according to the present invention in a plant orin a microorganism other than Micrococcus varians. To this end,nucleotides 154 to 228 of the sequence SEQ ID NO:2 can be cloned into anexpression vector downstream of a promoter or of a signal sequence andupstream of a terminator, while paying due regard to the reading frame,and the said vector can then be introduced into a plant, a bacterium ora yeast so as to increase their spectrum of inhibition towards certainbacteria, for example.

Use can be made of the signal sequence according to the invention byfusing nucleotides 88 to 153 of the sequence SEQ ID NO:2 to a gene ofinterest, while paying due regard to the reading frame, and by thencloning the whole into a Micrococcus varians expression vector, so as toenable to protein encoded by the said gene of interest to be expressedand secreted in Micrococcus varians, for example.

Nucleotides 88 to 228 of the sequence SEQ ID NO:2 can be cloned into aMicrococcus varians expression vector and introduced into another strainof Micrococcus varians so that this latter strain produces the variacinaccording the present invention.

In addition, the Micrococcus varians strain which contains, integratedinto its genome or by means of an expression vector, a DNA fragmentencoding the variacin according to the invention is also part of thesubject-matter of the present invention. In particular, the Micrococcusvarians strains which were deposited on 7 Jun. 1995, in accordance withthe Budapest Treating, in the Collection National de Cultures deMicroorganismes National collection of microorganism cultures!, INSTITUTPASTEUR, 25 Rue du Docteur Roux, F-75724 PARIS CEDEX 15, France, wherethey were given the deposition numbers CNCM I-1586 and CNCM I1587, arepart of the subject-matter of the present invention.

Micrococcus varians bacteria are Gram-positive, catalase-positive,aerobic bacteria which are permanently immobile. They are spherical inshape and are found in the form of tetrads which are arrangedirregularly.

Micrococcus varians colonies are coloured yellow on BHI medium. Theoptimum temperature for growing the said strains is 25°-37° C.

Strains CNCM I-1586 and CNCM I-1587, which are part of thesubject-matter of the present invention, metabolize both glucose andfructose. The CNCM I-1587 strain additionally metabolizes sucrose andfuranose.

In addition, strain CNCM I-1586 harbours two plasmids, of 4 and 12 kb,while strain CNCM I-1587 only harbours a single plasmid of 7 kb.

The culture supernatants of strains CNCM I-1586 and CNCM I-1587 exhibita relatively wide inhibition spectrum with regard to the growth of otherbacteria. The following may be included, by way of example, among thebacteria which are sensitive to the said supernatants: Lactococcuslactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp.bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillusdelbrueckii subsp. delbrueckii, Lactobacillus acidophilus, Lactobacillusjohnsonii, Lactobacillus plantarum, Lactobacillus saks, Lactobacilluscurvatus, Lauconostoc carnosum, Leuconostoc mesenteroides subsp.mesenteroides, Streptococcus thermophilus, Listeria monocytogenes,Enterococcus faecalis subsp. faecalis, the spores and vegetative cellsof Bacillus subtilis, Bacillus cereus, Bacillus polymyxa, Bacilluscirculans, Bacillus pumulus, and Bacillus liqueniformis, and theClostridia.

Preferably, in the process for preparing the variacin, a Micrococcusvarians strain which produces the said bacteriocin is cultivated, in amedium and under conditions which are favourable for growth, so as toobtain a culture medium containing from 10⁷ to 10¹¹ organisms of thesaid strain per ml, after which the resulting culture is centrifuged anda supernatant extract containing the said bacteriocin is obtained.

In order to produce this extract, the Micrococcus varians strainaccording to the present invention which produces the variacin, inparticular strain CNCM I-1586 or strain CNCM I-1587, can be cultured ina medium and under conditions which are favourable for the growth ofMicrococcus varians. To this end, cultivation can take place, inparticular, in BHI medium, at 25°-37° C. under aerobic conditions andwith shaking, until a concentration of 10⁷ -10¹¹ organisms per ml ofmedium is obtained, for example. The standard culture which is thusobtained is centrifuged at 4000-6000 g and the supernatant extractcontaining the said bacteriocin is collected.

The present invention also relates to the use of variacin, in particularin extract form, or the use of a Micrococcus varians strain whichproduces this bacteriocin, for preparing foodstuffs and cosmetics.

A culture of one of the said Micrococcus varians strains according tothe present invention may, in particular, be used in the fermentation ofmeat in order to prepare salami so as to combat contamination withClostridia, for example.

Variacin, in crude extract or purified form, may be used, when added tothe leaven which is obtained employing bacteria which are resistant tothe said variacin, in the preparation of cheeses, in particular cheesesof the mozzarella type, in order to avoid the holes which are producedby Bacillus polymixa whose spores survive the fermentation, and of thevariacin type in order to combat contamination with Listeriamonocytogenes, for example.

Variacin, in particular in crude extract or purified form, or one of thetwo stains, may be used as an additive or agent which is active againstpathogenic bacteria in the preparation of dessert mousses such aspasteurized custards, so as to combat the growth of spores such as ofClostridia and Bacillus cereus, or of bacterial strains such asListeria, for example.

In addition, variacin, in crude extract or purified form, or one of thetwo strains, may be used as an additive or agent which is active againstpathogenic bacteria in the preparation of cosmetics, such asmoisturizing creams or deodorants, so as to combat pathogenic bacteriaof the skin, for example.

ILLUSTRATIVE DESCRIPTION OF VARIACIN CHARACTERISTICS AND ACTIVITY

The variacin according to the present invention is characterized in moredetail below with the aid of various microbiological, biochemical andgenetic findings which illustrate its properties. The percentages aregiven by weight.

Unit of antibacterial activity--"agar well test"

Within the context of the present exposition, bacterial activity isdefined in terms of arbitrary units.

A supernatant of a standard culture of Micrococcus varians according tothe present invention, which supernatant is prepared, for example, underthe conditions described in Example 1, typically exhibits an activity of640 au/ml. Similarly, a concentrate of the said supernatant, whichconcentrate is prepared, for example, under the conditions described inExample 2, typically exhibits an activity of approximately 24000 au/ml.

The said agar well test is used to determine whether a sample stilldisplays bactericidal activity at a given dilution value.

In order to do this, 15 ml of MRS agar medium containing an indicatorstrain at a concentration of 10⁵ -10⁶ CFU/ml are inoculated into a Petridish. A strain which is sensitive to variacin, in this case the strainLactobacillus bulgaricus (YL 5) or the strain Lactobacillus helveticus(N 2), for example, is used as the indicator strain.

Holes of 5 mm in diameter are bored in the culture medium. The samplesof the supernatant or of the supernatant concentrate to be tested arepoured into the holes at the rate of 50 μl per hole. The dish ispreincubated at 4° C. for 2 h and then incubated overnight at either 30°C. or 37° C. depending on the indicator strain employed. Following theincubation, the indicator strain has grown and inhibition halos arevisible. The dilution value at which a sample no longer exhibitsbactericidal activity is the dilution value starting from which aninhibition halo is no longer discerned.

Inactivation by enzymes

The said agar well test is used to determine whether the variacin whichhas been isolated in accordance with the present invention isinactivated in the presence of a proteolytic enzyme or in the presenceof catalase.

In order to do this, enzyme is added, at the rate of 5 mg/ml, to aconcentrate of the culture supernatant described in Example 2. Theenzyme is allowed to act at the incubation temperature for 60 min beforethe sample is deposited in the agar well test well.

A control is prepared in parallel using the same concentrate at pH 7 andwithout the addition of enzyme. This control sample is incubated at 37°C. for 60 min before it is deposited in the agar well test well for thepurpose of comparing the inhibition halos obtained in the presence ofenzyme with the inhibition halo of the control. The diameter of thecontrol inhibition halo is 27 mm.

Table I below gives the results which were obtained with the enzymeswhich were tested using the indicator strain Lactobacillus helveticus (N2). In this table, as in Table II, the enzyme is designated by its type,the name of the supplier and the supplier's catalogue number. Thenumeral 0 indicates that there is no longer any halo, in other wordsthat the bactericidal activity of the variacin has been compromised byincubating the latter with the enzyme. The numeral 27 indicates thatthere is still a halo of 27 mm, corresponding to the full bactericidalactivity of the variacin.

                  TABLE I                                                         ______________________________________                                                         Incubation temperature                                                                      Inactivation                                   Enzymes          (°C.)  (mm)                                           ______________________________________                                        Catalase (SIGMA C-10)                                                                          30            27                                             Pronase E (SIGMA P-8038)                                                                       37            0                                              Proteinase K (MERCK 1000 144)                                                                  37            0                                              Ficin (SIGMA F-3266)                                                                           37            0                                              ______________________________________                                    

Table II below gives the results which were obtained with the enzymeswhich were tested using the indicator strain Lactobacillus bulgaricus(YL 5).

                  TABLE II                                                        ______________________________________                                                         Incubation temperature                                                                      Inactivation                                   Enzymes          (°C.)  (mm)                                           ______________________________________                                        Catalase (SIGMA C-10)                                                                          30            27                                             Pronase E (SIGMA P-8038)                                                                       37            0                                              Proteinase K (MERCK 1000 144)                                                                  37            0                                              Ficin (SIGMA F-3266)                                                                           37            0                                              ______________________________________                                    

The results given in Tables I and II show that all the proteolyticenzymes suppress the bactericidal activity of the variacin. Theseresults demonstrate the fact that variacin is proteinaceous in natureand that this proteinaceous moiety is involved in the bactericidalactivity.

The fact that catalase is not found to exert any influence on thebactericidal activity of the variacin also demonstrates that inhibitionof the growth of the two indicator strains is not due to theantibacterial activity of H₂ O₂, which is known to have a similaractivity to that of the bacteriocins, since H₂ O₂ would have beendegraded by the catalase.

Inhibition spectrum of the culture supernatant containing the variacin

The agar well test is used to determine whether the bactericidalactivity of the culture supernatant containing the variacin according tothe present invention exhibits an activity which is inhibitory to thegrowth of the different strains of spores and bacteria. The inhibitionspectrum of the supernatant is thus determined.

In order to carry out these assays, 15 ml of a standard medium, which isinoculated with 15 μl of a culture of the strain to be tested, whichculture was prepared during the preceding night, are poured into Petridishes so as to obtain a bacterial concentration of 10⁵ -10⁶ per ml ofstandard medium. The standard medium is the medium which is favourableto the growth of the strain to be tested.

Furthermore, when the strain to be tested has to grow from spores, 10⁵-10⁶ spores are inoculated per ml of covering medium.

A hole of 5 mm in diameter is bored in each Petri dish. A sample of 50μl of culture supernatant, as described in Example 1, is depositedtherein. The dishes are preincubated at 4° C. for 2 h and are thenincubated at a temperature which is favourable to the growth of thestrain under test for the time which is required for the strain to coverthe plate with a visible bacterial lawn.

The effect, or the degree of inhibition, herein the "agar wellincubation inhibition activity", is characterized by the diameter of theobserved inhibition halo. The inhibition halo is regarded as being verystrong (++++) if the halo exhibits a diameter of 18-28 mm, strong (+++)if the diameter if 10-17 mm, average (++) if the diameter is 5-9 mm,weak (+) if the diameter is 1-4 mm, and zero (-) if no halo is observed.

32 strains of lactic acid bacteria of different species and subspeciesare tested in this way and it is noted that none of them is resistant tothe supernatant. The detailed results of these tests are presented inTable III below. In Table III, as in the following tables, the straindesignation or strain no. indicated is the no. which is attributed to itin the Nestle collection (Address: NESTEC S.A., Centre de Recherche,Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland). The temperaturewhich is indicated is the incubation temperature during the test. Themedium which is indicated is the standard medium favourable to thegrowth of the stain to be tested.

                  TABLE III                                                       ______________________________________                                        Species         No.     T (°C.)                                                                        Media Inhibition                              ______________________________________                                        Lactococcus lactis                                                                            SL2     37      MRS   +++                                     Lactobacillus helveticus                                                                      N 2     37      MRS   +++                                                     N 258   37      MRS   +++                                                     N 262   37      MRS   +++                                                     N 271   37      MRS   +++                                                     LBL 4   37      MRS   ++++                                    Lactobacillus delbrueckii                                                                     N 9     37      MRS   ++                                      subsp. lactis   N 62    37      MRS   ++                                      Lactobacillus delbrueckii                                                                     LFi 1   37      MRS   +++                                     subsp. bulgaricus                                                                             LFi 5   37      MRS   +++                                                     YL 5    37      MRS   +++                                                     YL 18   37      MRS   +++                                     Lactobacillus delbrueckii                                                                     N 8     37      MRS   +++                                     subsp. delbrueckii                                                                            N 187   37      MRS   +++                                     Lactobacillus acidophilus                                                                     La 3    37      MRS   +++                                                     La 10   37      MRS   ++                                                      La 27   37      MRS   ++                                                      La 28   37      MRS   ++                                      Lactobacillus johnsonii                                                                       LA 1    30      MRS   ++                                      Lactobacillus plantarum                                                                       60      30      MRS   +                                                       3.4 RP  30      MRS   +                                       Lactobacillus sake                                                                            LSK     30      MRS   +                                       Lactobacillus curvatus                                                                        18      30      MRS   ++                                      Leuconostoc carnosum                                                                          LCA 3   37      M 17  ++++                                    Leuconostoc mesenteroides                                                                     A 74    30      MRS   +                                       subsp. mesenteroides                                                                          B 50    30      MRS   +                                       Streptococcus thermophilus                                                                    SFi 13  37      Elliber                                                                             ++                                                      SFi 16  37      Elliber                                                                             ++                                                      SFi 21  37      Elliber                                                                             ++                                                      SFi 21  37      Elliber                                                                             ++                                                      SFi 25  37      Elliber                                                                             ++                                                      ST 11   37      Elliber                                                                             ++                                                      ST 1    37      Elliber                                                                             ++                                      ______________________________________                                    

In Table III, it is noted that the inhibition spectrum of thesupernatant is broad in the sense that the degree of inhibition is ofabout the same level for the different species tested.

The inhibition spectrum of the supernatant of a culture producing thevariacin of the invention is also regarded as being broad in the sensethat it is not limited to species of lactic acid bacteria but extends toother species of Gram-positive bacteria, in particular to theundesirable or pathogenic bacteria Listeria innocua, Listeria welhia,Listeria monocytogenes, and to the spores of the Bacilli, for example,as is attested by the results presented in Table IV below.

                  TABLE IV                                                        ______________________________________                                        Species        No.      T (°C.)                                                                        Media Inhibition                              ______________________________________                                        Enterococcus faecalis                                                                        J        37      Elliber                                                                             ++                                      subsp. faecalis                                                               Enterococcus faectum                                                                         D 325    37      Elliber                                                                             ++                                                     MB 26    37      Elliber                                                                             ++                                      Listeria innocua                                                                             7        30      BHI   +++                                     Listeria monocytogenes                                                                       FSM 122  30      BHI   ++++                                    Listeria welhia                                                                              2        30      BHI   ++                                      Bacillus subtilis                                                                            A 1      30      BHI   +++                                     (spores and vegetative cells                                                                 A 2      30      BHI   ++                                                     A 3      30      BHI   ++                                                     A 4      30      BHI   ++                                                     A 13     30      BHI   +++                                     Bacillus subtilis                                                                            A 15     30      BHI   ++                                      (spores and vegetative cells                                                                 24       30      BHI   ++                                                     152      30      BHI   -                                       Bacillus cereus                                                                              C 1      30      BHI   ++                                      (spores and vegetative cells)                                                                C 2      30      BHI   ++                                                     C 5      30      BHI   ++                                                     C 6      30      BHI   ++                                                     79       30      BHI   ++++                                                   C 15     30      BHI   +                                       Bacillus amyloliquefaciens                                                                   226      30      BHI   ++++                                    Bacillus polymyxa                                                                            252      30      BHI   ++++                                    Bacillus liqueniformis                                                                       64       30      BHI   ++++                                    Bacillus stearothermophilus                                                                  10       30      BHI   -                                       Bacillus circulans                                                                           15       30      BHI   ++++                                    Bacillus pumulus                                                                             B 1      30      BHI   ++                                      (spores and vegetative cells)                                                                B 2      30      BHI   +++                                                    213      30      BHI   ++++                                    Clostridium botulinum                                                                        100003   45      DRMC  ++                                      (spores and vegetative cells)                                                                100006   45      DRMC  ++                                                     100019   45      DRMC  ++                                                     100023   45      DRMC  ++                                      Clostridium butyricum                                                                        102001   45      DRMC  ++                                      (spores and vegetative cells)                                                 Clostridium tyrobutyricum                                                                    107002   45      DRMC  +                                       spores and vegetative cells)                                                  Clostridium perfringens                                                                      103001   45      DRMC  +                                       (spores)                                                                      Clostridium sporogenes                                                                       104001   45      DRMC  +                                       (spores and vegetative cells)                                                 Clostridium acetobutilycum                                                                   106001   45      DRMC  +                                       (spores and vegetative cells)                                                 Clostridium    105001   45      DRMC  +                                       thermosaccharolyticum                                                         (spores and vegetative cells)                                                 Staphylococcus aurens                                                                        3        30      BHI   +                                                      15       30      BHI   +                                                      44       30      BHI   +                                                      60       30      BHI   ++                                      Staphylococcus xylosus                                                                       1        30      BHI   +                                       Staphylococcus simulans                                                                      1        30      BHI   +                                       Staphylococcus carnosus                                                                      1        30      BHI   ++                                                     14       30      BHI   ++                                      Staphylococcus saprophyticus                                                                 1        30      BHI   +                                                      15       30      BHI   +                                       Staphylococcus warneri                                                                       1        30      BHI   +                                       Staphylococcus cohnii                                                                        3        30      BHI   +                                       ______________________________________                                    

The results shown in Table VI make it possible, inter alia, to forecastattractive uses for this supernatant, or for the purified variacin, asan additive in the preparation of foodstuffs, in the role of an agentwhich is active against pathogenic bacteria, in particular againstClostridia in meat products, against Listeria monocytogenes in cheeses,against Bacillus cereus and Listeria in dessert mousses, or against theBacilli in fresh pastes or sauces for fresh pastes, from precisely whichbacteria, for example, the above strains originate.

Finally, variacin does not exert any inhibitory effect on the growth ofGram-negative bacteria as can be noted from the results shown in Table Vbelow.

                  TABLE V                                                         ______________________________________                                        Species        No.      T (°C.)                                                                        Media Inhibition                              ______________________________________                                        E. coli        1        30      BHI   -                                       Enterobacter chloacae                                                                        72       30      BHI   -                                       Salmonella anatum                                                                            XIV/20   30      BHI   -                                       Salmonella typhimurium                                                                       XIV/274  30      BHI   -                                       Pseudomonas fluorescens                                                                      3        30      BHI   -                                       ______________________________________                                    

Inhibition spectrum of a concentrate of the supernatant containingvariacin

The procedure is as described above except that the inhibitory effect onthe growth of different strains of spores and bacteria is determinedwhich is produced by a supernatant concentrate obtained as described inExample 2.

The same species and subspecies are tested as previously. The results ofthese tests are presented in Tables VI, VII and VIII below. The straindesignation or strain no. indicated is the no. which is attributed to itin the Nestle collection (Address: NESTEC S.A., Centre de Recherche,Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland). The temperaturewhich is indicated is the incubation temperature during the test. Themedium which is indicated is the standard medium favourable to thegrowth of the strain to be tested.

                  TABLE VI                                                        ______________________________________                                        Species         No.     T (°C.)                                                                        Media Inhibition                              ______________________________________                                        Lactococcus lactis                                                                            SL2     37      MRS   +++                                     Lactobacillus helveticus                                                                      N 2     37      MRS   ++++                                                    N 258   37      MRS   ++++                                                    N 262   37      MRS   ++++                                                    N 271   37      MRS   ++++                                                    LBL 4   37      MRS   ++++                                    Lactobacillus delbrueckii                                                                     LFi 1   37      MRS   ++++                                    subsp. bulgaricus                                                                             LFi 5   37      MRS   ++++                                                    YL 5    37      MRS   ++++                                                    YL 18   37      MRS   ++++                                    Lactobacillus delbrueckii                                                                     N 9     37      MRS   +++                                     subsp. lactis   N 62    37      MRS   +++                                     Lactobacillus delbrueckii                                                                     N 8     37      MRS   ++++                                    subsp. delbrueckii                                                                            N 187   37      MRS   ++++                                    Lactobacillus acidophilus                                                                     La 3    37      MRS   ++++                                                    La 10   37      MRS   ++++                                                    La 27   37      MRS   +++                                                     La 28   37      MRS   +++                                     Lactobacillus johnsonii                                                                       LA 1    30      MRS   +++                                     Lactobacillus plantarum                                                                       60      30      MRS   ++                                                      3.4 RP  30      MRS   ++                                      Lactobacillus sake                                                                            LSK     30      MRS   +++                                     Lactobacillus curvatus                                                                        18      30      MRS   +++                                     Leuconostoc carnosus                                                                          LCA 3   37      M 17  ++++                                    Leuconostoc mesenteroides                                                                     A 74    30      MRS   ++                                      subsp. mesenteroides                                                                          B 50    30      MRS   ++                                      Streptococcus thermophilus                                                                    SFi 13  37      Elliber                                                                             +++                                                     SFi 16  37      Elliber                                                                             +++                                                     SFi 21  37      Elliber                                                                             +++                                                     SFi 25  37      Elliber                                                                             +++                                                     ST 1    37      Elliber                                                                             +++                                                     ST 11   37      Elliber                                                                             +++                                     ______________________________________                                    

                  TABLE VII                                                       ______________________________________                                        Species        No.      T (°C.)                                                                        Media Inhibition                              ______________________________________                                        Enterococcus faecalis                                                                        J        37      Elliber                                                                             +++                                     subsp. faecalis                                                               Enterococcus faectum                                                                         D 325    37      Elliber                                                                             +++                                                    MB 26    37      Elliber                                                                             +++                                     Listeria innocua                                                                             7        30      BHI   ++++                                    Listeria monocytogenes                                                                       FSM 122  30      BHI   ++++                                    Listeria welhia                                                                              2        30      BHI   ++++                                    Bacillus subtilis                                                                            A 1      30      BHI   +++                                     (spores and vegetative cells)                                                                A 2      30      BHI   ++                                                     A 3      30      BHI   ++                                                     A 4      30      BHI   ++                                                     A 13     30      BHI   +++                                                    A 15     30      BHI   ++                                                     24       30      BHI   ++                                                     152      30      BHI   -                                       Bacillus cereus                                                                              C 1      30      BHI   ++                                      (spores and vegetative cells)                                                                C 2      30      BHI   ++                                                     C 5      30      BHI   ++                                                     C 6      30      BHI   ++                                                     79       30      BHI   ++++                                                   C 15     30      BHI   +                                       Bacillus amyloliquefaciens                                                                   226      30      BHI   ++++                                    Bacillus polymyxa                                                                            252      30      BHI   ++++                                    Bacillus liqueniformis                                                                       64       30      BHI   ++++                                    Bacillus stearothermophilus                                                                  10       30      BHI   -                                       Bacillus circulans                                                                           215      30      BHI   ++++                                    Bacillus pumulus                                                                             B 1      30      BHI   ++                                      (spores and vegetative cells)                                                                B 2      30      BHI   +++                                                    213      30      BHI   ++++                                    Clostridium butyricum                                                                        102001   45      DRMC  +++                                     (spores and vegetative cells)                                                 Clostridium perfringens                                                                      103001   45      DRMC  ++                                      (spores)                                                                      Clostridium tyrobutyricum                                                                    107002   45      DRMC  ++                                      (spores and vegetative cells)                                                 Clostridium sporogenes                                                                       104001   45      DRMC  ++                                      (spores and vegetative cells)                                                 Clostridium acetobutilycum                                                                   106001   45      DRMC  ++                                      (spores and vegetative cells)                                                 Clostridium    105001   45      DRMC  ++                                      thermosaccharolyticum                                                         (spores and vegetative cells)                                                 Clostridium botulinum                                                                        100003   45      DRMC  +++                                     (spores and vegetative cells)                                                                100006   45      DRMC  +++                                                    100019   45      DRMC  +++                                                    100023   45      DRMC  +++                                     Staphylococcus aureus                                                                        3        30      BHI   ++                                                     15       30      BHI   ++                                                     44       30      BHI   +                                                      60       30      BHI   +++                                     Staphylococcus xylosus                                                                       1        30      BHI   +                                       Staphylococcus simulans                                                                      1        30      BHI   +                                       Staphylococcus carnosus                                                                      1        30      BHI   +++                                                    14       30      BHI   +++                                     Staphylococcus saprophyticus                                                                 1        30      BHI   ++                                                     15       30      BHI   ++                                      Staphylococcus warneri                                                                       1        30      BHI   ++                                      Staphylococcus cohnii                                                                        3        30      BHI   ++                                      ______________________________________                                    

                  TABLE VIII                                                      ______________________________________                                        Species        No.      T (°C.)                                                                        Media Inhibition                              ______________________________________                                        E. coli        1        30      BHI   -                                       Enterobacter chloacae                                                                        72       30      BHI   -                                       Salmonella anatum                                                                            XIV/20   30      BHI   -                                       Salmonella typhimurium                                                                       XIV/274  30      BHI   -                                       Pseudomonas fluorescens                                                                      3        30      BHI   -                                       ______________________________________                                    

The results shown in Tables VI, VII and VIII demonstrate the increasedefficacy of the supernatant concentrate, as compared with thesupernatant, in inhibiting the growth of many of the strains tested.Inhibition spectra are observed for the same species and subspecies, butat a higher level of inhibition.

This suggests the preparation of a variacin supernatant concentrate, asdescribed in Example 2, and its use for combating pathogenic bacteria inthe preparation, for example, of foodstuffs and cosmetics.

Resistance to pH

The agar well test is used to determined whether the variacin which hasbeen isolated in accordance with the present invention is pH-dependent.

To this end, the agar is inoculated with an indicator strain, aspreviously described in the "agar well test". Lactobacillus helveticus(N 2) is used as the indicator strain.

The pH of an extract of the concentrate of the culture supernatantdescribed in Example 2 is adjusted to a pH of from 2 to 10 with 2N NaOHand/or 2N HCl, the extract is incubated at 37° C. for 60 min and the pHis then readjusted to 6-7 before a sample of the extract is deposited inthe agar well test well.

A control, using the same supernatant concentrate at pH 7, is set up inparallel and incubated at 37° C. for 60 min before the control sample isdeposited in the agar well test well so as to compare the inhibitionhalos of the test samples with the inhibition halo of the control. Thediameter of the inhibition halo of the control is 27 mm.

In Table IV, as in Tables X and XI, the numeral 27 indicates that thereis still a halo 27 mm, corresponding to the full bactericidal activityof the variacin.

                  TABLE IX                                                        ______________________________________                                               pH   Inhibition halos                                                  ______________________________________                                               2    27                                                                       4    27                                                                       6    27                                                                       8    27                                                                       10   27                                                                ______________________________________                                    

The results shown in the above table demonstrate that the bactericidalactivity of the variacin is not compromised. It is therefore possible toconclude that the bactericidal activity of variacin is not pH-dependent.

Resistance to heat

The agar well test is used to determine whether the variacin which hasbeen isolated in accordance with the present invention isheat-dependent.

To this end, the agar is inoculated with an indicator strain, aspreviously described in the agar well test. Lactobacillus helveticus (N2) is used as the indicator strain.

A concentrate extract of the culture supernatant, described in Example 2and adjusted to pH 7, is incubated at 100° C. for 15 to 60 min before asample of it is deposited in the agar well test well.

A control, obtained using the same supernatant concentrate at pH 7, isset up in parallel and incubated at 37° C. for 60 min. This enables theinhibition halos of the temperature test samples to be compared with theinhibition halo of the control. The diameter of the inhibition halo ofthe control is 27 mm.

                  TABLE X                                                         ______________________________________                                        Temperature (°C.)                                                                 Incubation time (min)                                                                        Inhibition halos (mm)                               ______________________________________                                        100        15             27                                                  100        30             27                                                  100        60             27                                                  ______________________________________                                    

These results demonstrate that variacin is not heat-dependent. Thus, thebactericidal activity of variacin is not compromised even after a 60 minincubation at 100° C.

The resistance of variacin to heat is a biochemical characteristic whichis of great important in relation to using variacin in the preparationof foodstuffs and cosmetics. Thus, variacin can be used, in particularin crude extract or purified form, in the preparation of pasteurizedfoodstuffs, so as to combat the growth of spores, such as, for example,the Bacilli, which are resistant to heat.

Resistance to heat and to pH

In addition, the stability of variacin is tested when combining pH andheat.

To this end, the agar is inoculated with an indicator strain, aspreviously described in the "agar well test". Lactobacillus helveticus(N 2) is used as the indicator strain.

The culture supernatant concentrate extract described in Example 2 isadjusted to pH 4 or 7 with 2N HCl and/or 2N NaOH and incubated at 115°C. for 20 min; the pH of the extract is then readjusted to pH 6-7 beforea sample of it is deposited in the agar well test well.

A control, obtained at pH 7, is set up in parallel at 37° C. for 20 min,before depositing the control sample in the agar well test well so as tocompare the inhibition halos of the test samples with the inhibitionhalo of the control. The diameter of the inhibition halo of the controlis 27 mm.

                  TABLE XI                                                        ______________________________________                                              Incubation time                                                                           Incubation temperature                                                                       Inhibition halo                              pH    (min)       (°C.)   (mm)                                         ______________________________________                                        4     20          115            27                                           7     20          115            27                                           ______________________________________                                    

The results given in the above table demonstrate that the bactericidalactivity of variacin is not compromised at pH 4 or 7, combined with anelevated temperature.

Purification of variacin

4 l of BHI medium are inoculated with a culture of Micrococcus varians,which culture produces the variacin according to the present invention.This standard culture is incubated at 30° C. overnight under aerobicconditions and while shaking, after which it is centrifuged at 5000 g soas to collect the supernatant in a recipient vessel, to which 72 g ofXAD-7 resin (Amberlite (R)) are added.

The mixture is stirred at 25° C. for 30 min in order to facilitateadhesion of the variacin molecules to the resin, and the whole is thentransferred onto a sintered glass where the supernatant is filtered invacuo.

The resin is washed successively in 3 buffers containing 20 mM sodiumcitrate, pH 4, and isopropanol. The first contains 10% isopropanol, thesecond buffer contains 15% isopropanol, and the third buffer contains20% isopropanol.

The resin is transferred onto a column and the variacin is eluted with700 ml of buffer containing 20 mM sodium citrate, pH 4, and 25%isopropanol The bactericidal activity of the variacin is monitored usingthe agar well test as described previously.

The active fractions are mixed and the isopropanol is evaporated. A 5 mlS-Resource column for FPLC (Pharmacia) is prepared by equilibrating itwith 20 mM sodium citrate buffer. The evaporated mixture of activefractions is loaded onto this column and the contents of the column arethen eluted with an NaCl buffer having a gradient of from 100 mM to 400mM.

Fractions are collected and the bactericidal activity of the variacin,which has thus been purified, is checked using the agar well test.

Sequencing the variacin

The N-terminal part of the variacin purified from strains CNCM I-1586and CNCM I-1587 is sequenced using an Applied Biosystems 4774 automaticsequencer.

This reveals the peptide sequence of 5 amino acids whose sequence isidentical to that for the N-terminal part of the sequence SEQ ID NO:1,described in the sequence list below.

It was not possible to demonstrate the presence of a peptide of morethan 5 amino acids during the sequencing.

In addition, variacin which has been purified from strains CNCM I-1586and CNCM I-1587 is hydrolysed with 6N HCl for 10 min. 3 peptides areobtained which are isolated in the usual manner by HPLC. It was onlypossible to sequence one of the three peptides which were isolated sincethe other two most probably contain peptide modifications. The sequenceof the said peptide which was isolated in this way is identical to thatcomprising amino acids 19 to 22 of the sequence SEQ ID NO:1, describedin the sequence list below.

Finally, a fraction containing the variacin which has been purified fromstrains CNCM I-1586 and CNCM I-1587 is subjected to mass spectrometryand the variacin is found to have a molecular weight of 2659 daltons.

Homology

Homology was demonstrated between the sequence of lacticin 481 fromLactococcus lactis and that of the variacin according to the presentinvention. This homology relates, in particular, to the sequences of theN-terminal part of the two bacteriocins. Thus, amino acids 1 to 5 of theN-terminal sequence of variacin are identical to amino acids 3 to 7 ofthe sequence SEQ ID NO:8 of lacticin 481, described in the sequence listbelow. Nevertheless, it is only a matter of partial homology and not ofidentity.

In addition, secreted lacticin 481 is shown by mass spectrometry to havea molecular weight of 2900 daltons, whereas the molecular weight ofsecreted variacin is 2659 daltons, as we have previously seen.

When the strain Lactococcus lactis which produces lacticin 481 isinoculated in the presence of a variacin extract, as previouslydescribed in the inhibition spectrum test, the growth of the said strainis seen to be inhibited. The strain of Lactococcus lactis which produceslacticin 481 is immune to its own bacteriocin, lacticin 481, but is notimmune to the variacin which is produced by either of the twoMicrococcus varians strains according to the present invention. Theseresults, which were obtained in the previously described inhibitionspectrum test, confirm that these two bacteriocins are different.

The above genetic findings demonstrate that while lacticin 481 andvariacin exhibit sequence homologies, their sequences are not identical.The biochemical findings, as well as the microbiological findings,confirm that lacticin 481 and variacin are two different bacteriocins.

Sequencing the variacin gene

The degenerate nucleotide sequence SEQ ID NO:4, which is described inthe sequence list below and which corresponds to the C-terminal part ofthe peptide of the previously sequenced variacin, is constructed in aconventional manner. The mixture of SEQ ID NO:4 sequences is thenrendered radioactive by the action of T4 polynucleotide kinase.

A preparation of chromosomal DNA is made from strains CNCM I-1586 andCNCM I-1587 in a conventional manner. The said DNA preparation isdigested with SalI, SacI, SphI and BamHI in accordance with therecommendations of the enzyme suppliers. 2 μg of the digestion productare then run on an agarose gel. The DNA on the gel is washed with 250 mMHCl and the migration product is then transferred, in alkaline medium,from the gel onto a "Zetaprobe" (Biorad) membrane. The Zetaprobemembrane is then prehybridized at a temperature of 55° C., whichtemperature is lowered by 5° C. every 2 h down to a temperature of 40°C., in a medium comprising 6×SSC, 1% SDS and 0.25% skimmed milk. Thismembrane is hybridized with the degenerate radioactive probe exhibitingthe sequence SEQ ID NO:4 in the previous hybridization medium and underthe same temperature conditions. It is then left to incubate at 40° C.for 4 hours, after which the membrane is washed at 40° C. in 6×SSC.Finally, it is exposed on an autoradiography film at -80° C. for 16 h.

The hybridization demonstrates a variety of migration bands: a SalI bandof 7 kb, a SacI band of 1.4 kb, a BamHI band of 1.8 kb and an SphI bandof greater than 15 kb.

5 μg of genomic DNA from strain CNCM I-1586 is then digested with therestriction enzyme BamHI and a fragment of 1.6-2 kb is separated byagarose gel chromatography followed by elution of that part of the gelcontaining the fragment. The eluted DNA fragment is ligated to thevector pK 19 (Gene, 56 (1987) 309-312), which has previously beendigested with BamHI and then treated with calf intestinal phosphatase(Boehringer Mannheim, part No. 713023).

The Escherichia coli strain BZ 234 (Biozentrum collection--University ofBasle, Switzerland), which has previously been rendered competent, isthen transformed, in a conventional manner, with the ligation medium.The clones containing the insert are identified on agar medium which issupplemented with 50 μg/ml kanamycin, 60 ng/ml IPTG (BoehringerMannheim, part No. 724815) and 300 ng/ml X-gal (Boehringer Mannheim,part No. 651745) and which is incubated at 37° C. for 16 h.

The white colonies, which normally contain an insert, are picked outinto 96-well microtitre plates. Each white colony is picked out into oneof the said wells, with each well containing 150 μl of LB mediumsupplemented with 50 μg/ml kanamycin, and incubated at 37° C. for 20 hin order to produce mini cultures.

Two primers of opposite orientation are prepared because the orientationof the gene in vector pK 19 is not shown. To this end, the said primersare constructed by assembling them from a nucleotide fragment exhibitingthe sequence SEQ ID NO:5, partially encoding lacticin 481, which islinked to one or other of the universal probes of the pUC vectors, whichprobes exhibit the sequence SEQ ID NO:6 or the sequence SEQ ID NO:7.

1 μl from each well is mixed with 100 pmol of one of the said primers, 6μl of 2 mM dNTPs and 2.5 μl of Taq buffer (P.H. Stehelin & Cie AG, cat.no. TP05b), and the whole is covered with a drop of Dyna-wax (FinnzymesOy, 02201 Espoo, Finland) and heated at 98° C. for 10 min so as to lysethe bacteria; the PCT is then carried out.

The positive clones then give a band of 800 bp on an electrophoresisgel.

The positive clones are selected in this way and the plasmid DNA ofthese clones is extracted; the DNA fragment which is cloned into the pK19 vector is then sequenced by the dideoxynucleotide method using asequencing kit (Pharmacia Biotech, part No. 27-1682-01) and universalprimers, followed by specific primers which are based on the sequencethus obtained.

This results in a nucleotide sequence, SEQ ID NO:2, which is describedin the sequence list below, being obtained. The said nucleotide sequenceencodes the sequence SEQ ID NO:1, which corresponds to the amino acidsequence of variacin, prior to maturation.

Variants of the protein conserving the bactericidal activity

Variants of the protein having the amino acid sequence SEQ ID NO:1 arecreated. To this end, the encoding DNA sequence of the variacin withoutthe peptide signal (recombinant vector pK19 above) is inserted into thepolyclonal site of plasmid pKK232-8 (Pharmacia, UK). Chemicalmutagenesis with hydroxylamine are performed on the expression vectoraccording to the process described by Yoast et al. (Applied and Env.Micro., 60, 1221-1226, 1994). Other methods could also be used, such asthe method described by Dunn et al. (Protein Engineering, 2, 283-291,1988) dealing with the creation of precise mutations.

The mutagenized vectors are transferred into competent E. coli BZ 234and transformants are selected.

Transformants are isolated and cultivated. Supernatants are prepared andconcentrated according to Example 2.

Each supernatant is then screened according to the "agar well test"described above.

Results show that some supernatants provide an inhibition activityagainst Lactobacillus, Lactococcus, Streptococcus, Enterococcus,Listeria, Bacillus, Clostridia and/or Staphylococcus, for example.

DNA analysis of vectors expressing the bacteriocin show that somebacteriocins present a DNA sequence which is different to the encodingsequence SEQ ID NO:2. The amino acid sequence of these variants aregenerally different from 1 to 4 amino acids.

EXAMPLES

The examples below are presented by way of illustrating the process forproducing, and the uses of, the bacteriocin according to the presentinvention. The percentages in the examples are given by weight unlessotherwise indicated.

EXAMPLE 1

BHI culture medium is inoculated, and a culture containing organisms ofthe Micrococcus varians strain is added to it. The whole is incubatedovernight at 30° C. with shaking and under aerobic conditions, afterwhich the medium contains 10⁸ organisms of the strain per ml. Thestandard culture thus obtained is centrifuged. The standard supernatantis collected.

EXAMPLE 2

The supernatant concentrate is obtained from 750 ml of the saidsupernatant, which has been obtained as described in Example 1 and towhich is added 15 g of XAD-7 resin (Amberlite (R)). The mixture isshaken at 4° C. for 60 min and is then filtered through a No. 604Schleicher & Schuell (Germany) filter. The filter is then washed with 1%sodium citrate in order to elute all the non-adsorbed proteins. Theresin is isolated and transferred into a recipient vessel containingsodium citrate and the whole is shaken for 2 min. The resin, togetherwith the sodium citrate, is transferred into a column and the sodiumcitrate is eluted with 50% acetonitrile and 0.1% TFA. The eluate isevaporated and the residue is resuspended in 50 mM phosphate buffer atpH 6.8.

EXAMPLE 3

10 liters of a culture of the Micrococcus varians strain are produced inBHI medium overnight at 30° C. with shaking and under aerobicconditions. 200 g of XAD-7 resin (Amberlite) are then added directly tothe culture and the whole is shaken gently at 4° C. for 1 h. The mixtureis then filtered through a No. 604 Schleicher & Schuell (Germany)filter, and the resin which is retained on the filter is then washedwith 10 liters of a 50 mM acetic acid, pH 5.2, solution in order toremove the bacteria. After that, 450 ml of a solution containing 100%ethanol and 20 mM ammonium acetate are added to the resin and the wholeis filtered in order to remove the resin; the filtrate is thenlyophilized in order to obtain a powder containing the variacinaccording to the invention, which can be used in the foodstuff industry.

The bactericidal activity of this powder, which has been previouslydiluted in water, is then determined by means of the previouslydescribed agar well test. This powder possesses at least 10⁵ au/gpowder.

Finally, the above powder is added, at the rate of 0.5 g/kg, to a meatfoam while it is being prepared in a traditional manner. This results ina meat foam, each g of which contains 50 au of bacteriocins which areable to completely inhibit the development of pathogenic bacteria, inparticular Clostridia.

EXAMPLE 4

This example relates to the preparation of a moisturizing cream for skincare containing the powder described in Example 3 at the rate of 0.05g/kg, that is, therefore, variacin, which is capable of inhibiting thedevelopment of undesirable bacteria on the skin, in particularStaphylococcus aureus and Streptococcus pyogenes, at the rate of 5 au/g.

In order to prepare this emulsion, the components of lipid phase A aremixed and heated at 75° C. Aqueous phase B is prepared and also heatedat 75° C.; lipid phase A is then added, while mixing slowly, and, afterthat, the whole is cooled, under slow mixing, down to ambienttemperature, that is approximately 25° C. The C constituents are addedslowly, at this temperature, in the order of the formula.

    ______________________________________                                                                    %                                                 ______________________________________                                        Lipid phase A                                                                 Peg-6 stearate, glycerate and peg-20-cethyl ether                                                           15                                              (peg: polyethylene glycol)                                                    Liquid paraffin               5                                               Wheat germ oil stabilized with 0.1% phenylindane                                                            3                                               (antioxidant) and 1% soybean phospholipid (see EP94109355.1)                  Sweet-almond oils             2                                               Cetyl alcohol                 1                                               Isostearyl isostearate        2                                               2-Octyldodecyl myristate      1                                               Lanolin wax                   1                                               Aqueous phase B                                                               Methylisothiazoline           0.1                                             Demineralized water           59.6                                            Human placenta protein        2                                               C Additives                                                                   Propylene glycol and calendula extract                                                                      2                                               50% soluble collagen in demineralized water                                                                 5.8                                             Perfume                       0.3                                             2.5% bacteriocin powder in accordance with Ex. 3 in                                                         0.2                                             demineralized water                                                           ______________________________________                                    

EXAMPLE 5

The bacteriocin powder described in Example 3 is added to a mouthwash atthe rate of 0.5 g/kg. This mouthwash is consequently capable ofinhibiting the development of pathogenic bacteria, in particularStreptococcus sorbrinae, Streptococcus sanguis, Streptococcus mutans andActinomyces viscosus, in the buccal cavity.

EXAMPLE 6

A solution comprising the bacteriocin powder of Example 3, which isdiluted in water at the rate of 1%, is sprayed onto a foodstuff which isintended to be sterilized in order to prevent post-contamination duringpackaging.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: MICROCOCCUS VARIANS                                             (C) INDIVIDUAL ISOLATE: TWO CLONES CNCM I-1586 and CNCM                       I-1587                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GlySerGlyValIleProThrIleSerHisGluCysHisMetAsnSer                              151015                                                                        PheGlnPheValPheThrCysCysSer                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 278 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: MICROCOCCUS VARIANS                                             (C) INDIVIDUAL ISOLATE: TWO CLONES CNCM I-1586 and CNCM                       I-1587                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 88..228                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 88..153                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 154..228                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GTTGAAAATCATCCGGAAGGATGATTCTGTGTTCATGCGAGGCCACCCGGAATCGCGGCA60                GCCACACCCACTGAGAGGACTAGAACAATGACGAACGCATTTCAGGCACTG111                        MetThrAsnAlaPheGlnAlaLeu                                                      22-20-15                                                                      GACGAAGTCACGGACGCCGAGCTCGACGCCATCCTTGGCGGGGGCAGT159                           AspGluValThrAspAlaGluLeuAspAlaIleLeuGlyGlyGlySer                              10-51                                                                         GGTGTTATTCCCACGATCAGCCACGAGTGCCACATGAACTCCTTCCAG207                           GlyValIleProThrIleSerHisGluCysHisMetAsnSerPheGln                              51015                                                                         TTCGTGTTCACCTGCTGCTCCTGAGAAACTCCTCCGATGCTCAGAGGGCCG258                        PheValPheThrCysCysSer                                                         2025                                                                          CGCTAGGAAAATCTAGTAAG278                                                       (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetThrAsnAlaPheGlnAlaLeuAspGluValThrAspAlaGluLeu                              22-20-15-10                                                                   AspAlaIleLeuGlyGlyGlySerGlyValIleProThrIleSerHis                              51510                                                                         GluCysHisMetAsnSerPheGlnPheValPheThrCysCysSer                                 152025                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "oligonucleotide"                                    (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TGGCARTTYGTNTTYACNTGYTG23                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "oligonucleotide"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GGNTCNGGNGTNATHCAYACNATHTCNCAYGARTGYAAYATGAA44                                (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "oligonucleotide"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ATAACAATTTCACACAGG18                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "oligonucleotide"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GGTTTTCCCAGTCACGAC18                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LysGlyGlySerGlyValIle                                                         15                                                                            __________________________________________________________________________

We claim:
 1. An isolated peptide of amino acid sequence minus 22 tominus 1 of SEQ ID NO:3.